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Abstract

We present a study characterizing the properties of femtosecond laser nanosurgery applied to individual axons in live Caenorhabditis elegans (C. elegans) using nano-Joule laser pulses at 1 kHz repetition rate. Emphasis is placed on the characterization of the damage threshold, the extent of damage, and the statistical rates of axonal recovery as a function of laser parameters. The ablation threshold decreases with increasing number of pulses applied during nanoaxotomy. This dependency suggests the existence of an incubation effect. In terms of extent of damage, the energy per pulse is found to be a more critical parameter than the number of pulses. Axonal recovery improves when surgery is performed using a large number of low energy pulses.

A. Waller, “Experiments on the section of glossopharyngeal and hypoglossal nerves of the frog and observations of the alternatives produced thereby in the structure of their primitive fibers,” Philos Trans R Soc Lond Biol. 140, 423 (1850).
[Crossref]

1973 (1)

1850 (1)

A. Waller, “Experiments on the section of glossopharyngeal and hypoglossal nerves of the frog and observations of the alternatives produced thereby in the structure of their primitive fibers,” Philos Trans R Soc Lond Biol. 140, 423 (1850).
[Crossref]

Waller, A.

A. Waller, “Experiments on the section of glossopharyngeal and hypoglossal nerves of the frog and observations of the alternatives produced thereby in the structure of their primitive fibers,” Philos Trans R Soc Lond Biol. 140, 423 (1850).
[Crossref]

Philos Trans R Soc Lond Biol. (1)

A. Waller, “Experiments on the section of glossopharyngeal and hypoglossal nerves of the frog and observations of the alternatives produced thereby in the structure of their primitive fibers,” Philos Trans R Soc Lond Biol. 140, 423 (1850).
[Crossref]

Figures (10)

(a) Sketch of all GABA-ergic neurons in C. elegans (head to the right). The 13 VDs and the 6 DDs are the motor neurons. The insert shows myosin filaments underlying an axon [17]. (b) Sketch of soft touch neurons in C. elegans (head to the left) [18]. ALM and PLM come in pairs; only one of each is accessible during surgery.

Fluorescence images of an axon of a GFP-labeled PLML neuron (a) before axotomy, (b) right after the axotomy at mid-body (100 pulses of 1.9 J/cm2), and (c) 12 hours after the axotomy showing regrowth of the severed axon and reconnection to its distal end.

Images of regrown ALML axon 72 hours after axotomy on a youg adult worm (300 pulses of 1.9 J/cm2) at three different focal depths. The first two images show the connection to the degenerated distal end and the third image shows how the process continues to regrow most probably in search of a healthier connection. (The distal and proximal ends are respectively on the upper and lower parts of the pictures).

Fluorescence images of photobleaching at 1.3 J/cm2, 25 pulses on CZ5062 , (a) before laser exposure, (b) right after exposure, and (c) 2 minutes after exposure. The irradiated spot loses its signal due to photobleaching but remains intact. The axon recovers its luminescence within 2 minutes by the GFP diffusion within the cytoplasm.

Fluorescence images of the extent of damage induced by fs-laser ablation using 800 pulses at 1.7 J/cm2. (a) before ablation, (b) right after ablation with GFP spilling in the created cavity, and (c) 3 minutes after ablation. The dark region in muscle fibers shows the extent of damage to surrounding tissue. The diameter of this area is approximately equal to the distance between the ends of the cut axon.

Ablation thresholds measured as a function of number of pulses. Solid line shows the logarithmic fit following Eq. (2). The corresponding fluences (energy per area) and irradiances are included in the right side axis assuming a theoretical spot size of 620 nm and a pulse duration of 430 fs. The error bars indicate the variance of the threshold measurements on 10 axons for different pulse trains.

Table 2. Statistics of axonal recovery of touch neurons and survival rate of worms after laser axotomy performed using different pulse energies and total number of pulses. Relative rates refer to the number of worms fulfilling the requirement of the previous column.

Free electrons participate in chemical reactions to form destructive reactive oxygen species and lead to breaking of chemical bonds.

Since thermalization of the plasma occurs faster than the acoustic relaxation time, confinement of thermal stresses leads to formation of nano-scale transient bubbles.

The damage is created by the extremely hot plasma and the accompanying shock wave and cavitation bubble.

Table 2.

Statistics of axonal recovery of touch neurons and survival rate of worms after laser axotomy performed using different pulse energies and total number of pulses. Relative rates refer to the number of worms fulfilling the requirement of the previous column.

Free electrons participate in chemical reactions to form destructive reactive oxygen species and lead to breaking of chemical bonds.

Since thermalization of the plasma occurs faster than the acoustic relaxation time, confinement of thermal stresses leads to formation of nano-scale transient bubbles.

The damage is created by the extremely hot plasma and the accompanying shock wave and cavitation bubble.

Table 2.

Statistics of axonal recovery of touch neurons and survival rate of worms after laser axotomy performed using different pulse energies and total number of pulses. Relative rates refer to the number of worms fulfilling the requirement of the previous column.